Structural features of reverse transcriptase (RT) that influence fidelity of reverse transcription in vivo are not well defined. We identified Y586F as a mutation in the MLV RT ribonuclease H (RNase H) primer grip subdomain that increased the mutation rate approximately fivefold-to date, the largest reported increase for a substitution mutation in the in vivo retroviral mutation rate. The Y586F mutation increased the frequency of substitutions 17-fold within 18 nt of adenine-thymine tracts (A-tracts), which induce DNA bending. These results suggest that when wild-type RT encounters irregular template-primer conformations such as those induced by A-tracts, the Y586 residue and the RNase H primer grip domain facilitate a template-primer conformation that is necessary for maintaining high fidelity of DNA synthesis. To further determine the effects of MLV RNase H primer grip on replication fidelity and viral replication, we performed additional mutational analysis. The results indicated that mutations in the RNase H primer grip significantly increased the frequency of deletions between the primer-binding site (PBS) and sequences downstream of the PBS. Quantitative real-time PCR analysis indicated that the mutants RTs were substantially inefficient in plus-strand DNA transfer relative to wild-type RT. These results indicate that the MLV RNase H primer grip is an important determinant of the in vivo fidelity of DNA synthesis. We plan to determine the role of several other structural determinants of RT that might influence fidelity of reverse transcription. In addition, we are testing the error catastrophe hypothesis by determining the effects of mutator RTs and mutagenic nucleoside analogs on replication and evolution of MLV and HIV-1.